Cellular Oncogenes

We made it to the 70s

v-srcc-srcproto-oncogene oncogene

Viral oncogenes paved the way

v-srcc-srcproto-oncogene oncogene

Viral oncogenes paved the way

The concept:-Viruses kidnap a normal proto-oncogene-During the “kidnapping”, the mutated proto-oncogene became an oncogene-A new viral infection inserted an oncogene into the recipient, leading to cancer

At the time, there wereno known viral tumors in humans

However….

We ultimately want to know the cause of human diseases like colon cancer,

rather than curing chicken sarcomas

http://www.clevelandclinic.org/registries/inherited/fap.htm

One oncogene mutated in the progression to malignancy is K-ras

Lodish et al. Fig. 24-6

How did we get there?

Bob Weinberg long before he wrote our textbook

Onto the stage stepped a bright and ambitiousyoung assistant professor with a crazy idea

But how can we identify oncogenes that are not viral oncogenes?

But how can we find oncogenes that are not viral oncogenes?

Add DNA from carcinogen-treated cell?

First you need a way to get DNA into cells

How would you do that?

DNA can be “transfected” into cells using Calcium Phosphate

As you know, Oncogenes relieve contact inhibition.This can be visualized in culture

by “focus formation”

Transfect with oncogene

J Virol 2000 74:1008-13 Yoshioka et al.

J Biol Chem 2002 277:10813-23 Fiordalisi et al.

One can then test if the cells induce tumors

But it’s a crazy idea

1. you need to find the right needle ina very large haystack

2.What if you need to find two or more needles??

Bob WeinbergChiaho Shih

Bob WeinbergChiaho Shih

Maybe it wasn’t So crazy after all!

Soon everybody was trying it!

DNA from chemically treated mouse cells can transform normal mouse cells

Without a virus: cells can be transformed

a focus after transfection cells from the focus cells near the focus

It’s time to move on- the 80s

Transfect DNA from human cancer cell lines

Human Cancer Cell Line

Could this work for human tumors?

Bob WeinbergChiaho Shih

Human Cancer Cell Line

Somewhere here, among the normal mouse genes, we have a human oncogene

Low transformation rates suggest that we are dealing with a single oncogene

Figure 4.8 The Biology of Cancer (© Garland Science 2007)

How to find a needle in a haystack

Lodish et al. Fig. 24-4

How to find a needle in a haystack?

Generate a bacteriophage genomic library

Searching for the one human gene among many

mouse genes

An Oncogene is Cloned From a Human Tumor !!

The Race is ON

December 1981Wigler lab

Barbacid lab

Weinberg lab

But what does it encode?

The cloned gene is ~25,000 bp

In 1981 there was no way to sequence 25 kilobases

The cloned gene is ~25,000 bp

In 1981 there was no way to sequence 25 kilobases

Luis ParadaBob Weinberg

Let’s try a long-shot shortcut--what if the cellular oncogene is one of the known viral oncogenes?

Its very unlikely- 14 v-oncogenes, ~30,000 human genes

Use Southern blot analysis to look for one human gene in the otherwise mouse genome

What probe should we use ?

Der et al. PNAS 82Channing Der, now at UNC

mouse RAS

human RAS

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Just go through the viral oncogenes one by one

Der et al. PNAS 82Channing Der, UNC

mouse RAS

human RAS

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Mouse cells transformed by a human oncogene

The transforming oncogene is RasCellular oncogenes = Viral oncogenes

v-rasc-ras

(cellular ) (viral)

Proto-oncogene Oncogene

VirusesCarcinogens

Random mutations

But what does ras do in the cell??

But what does ras do in the cell??

What do we want to know?

But what does ras do in the cell??

Where does it do its work?

Ras is postranslationally modifiedby addition of a lipid

--where will that put it?

Lipid modificationtargets Ras to the plasma membrane

Farnesyltransferase inhibitorsoffer a way of reducing Ras activity

Clinicians then tried them in a variety of tumorsWith activated Ras involvement

Examples of Phase II trials:Leukemias (esp. AML/CML):Metastatic breast cancer(with capecitabine):Pancreatic cancer (with gemcitabine):Ovarian cancer (with current 2 drug combo): Neuroblastoma and Small cell lung cancer (with Taxol):

TipifarnibOr Lonafarnib

Sadly, it was largely an epic fail

Examples of Phase II trials:Leukemias (esp. AML/CML)May have some efficacyMetastatic breast cancer(with capecitabine): no significant improvementPancreatic cancer (with gemcitabine): no improvementOvarian cancer (with current 2 drug combo): no effectNeuroblastoma and Small cell lung cancer (with Taxol): Discontinued.

TipifarnibOr Lonafarnib

What else could we ask about Ras?

Lodish et al. Fig. 20-5

Scientists found Ras binds GTPIs it a new type of kinase?

Lodish et al. Fig. 20-5

Ras is an enzyme and hydolyzes GTP

Lodish et al. Fig. 20-5

Does this remind youof another well studied signaling pathway

that was one of the first identified?

Nobel Prize.org

2012 Nobel Prize in Chemistry!

Lodish et al. Fig. 20-5

This allows Ras to act as a molecular switch

The activity of Ras is regulated byGEFs and GAPs

How is oncogenic ras different from the normal proto-oncogene?

A new race starts

How is oncogenic ras different from the normal proto-oncogene?

How is oncogenic ras different from the normal proto-oncogene?

Table 4.2 The Biology of Cancer (© Garland Science 2007)

Ras is a key player in >50% of human tumors!

Table 4.2 The Biology of Cancer (© Garland Science 2007)

A note for the future—Ras is mutated in <5% of human breast cancers—we’ll see why later

The G12V mutation prevents endogenous and GAP-stimulated GTPase activity

Lodish et al. Fig. 20-5

G12V

Thus the Ras oncogene is constitutively activei.e. stuck in the ON state!

How does Ras act in our body, in vivo ?

Why would constitutively active Ras lead to cancer ?

From cell culture to model organisms